Panax ginseng C.A. Meyer contains large amounts of useful substances, such as ginsenosides, polyacetylene compounds, polyphenol compounds, polysaccharides containing proteins with host defense functions, polysaccharides having anticomplementary activity, and acidic polysaccharides. However, it is difficult to cultivate and may cause problems associated with pesticide contamination, environmental destruction, etc. In addition, it is very expensive, because it must be cultivated for at least 4 years in order to use the root thereof for medicinal purposes, and thus much manpower and cost are required.
For this reason, studies on methods of using bioengineering methods to produce large amounts of ginseng cells in vitro or to produce ginseng adventitious roots, hairy roots and the like in large amounts have been conducted. It was reported that the growth rate of cell mass (called callus) obtained by culturing ginseng cells in vitro using such cell culture methods is higher than that of ginseng plants obtained in fields (Korean Patent Registration 10-0333559) and that the saponin content of cultured ginseng cells is not significantly lower than that of ginseng roots (Asaka et al., Plant Med., 59:345, 1993).
Accordingly, materials obtained by culturing ginseng adventitious roots (ginseng or true wild ginseng-Korea Forest Service, CBN Biotech, Neobio, KT&G Research Institute, Microplants Bioscience & Biotechnology, etc.) or ginseng cells (Nitto Denko, Japan, etc.) are being used as raw materials for foods and cosmetics (Korean Patent Registration 10-0601903, Korean Patent Registration 10-0637342, Korean Patent Publication 10-2004-0014584). Particularly, true wild ginseng is rare and very expensive, studies focused on culturing the adventitious roots and seedlings thereof to produce ginseng products in large amounts have been actively conducted in various companies and research institutes (Korean Patent Publication 10-2005-0078372).
When culturing herbaceous plants such as ginseng, true wild ginseng and the like, the tissue to be used as a culture material is the root, that is, the storage root. The storage root tissue is a part that is buried in soil for a long period of time to form various relationships with soil microorganisms while absorbing water and inorganic nutrients in the soil during the life thereof. In order to use the storage root tissue in plant cell or tissue culture, sterilization of the tissue is required. However, there are many reports of difficulties in removing microorganisms from the root tissue by surface sterilization because high concentration of sterilizing solution would destroy the tissue and low concentration of the sterilizing solution would cause contamination of the tissue with various fungi and bacteria. This contamination phenomenon becomes severe, particularly in the case of wild-cultivated ginseng and true wild ginseng, which have grown in soil for a long period of time (Korean Patent Registration 10-0478213; Teng, W. L. et al., Plant Cell Tissue Organ Cult., 68:233, 2002).
Also, in order to produce large amounts of cells of ginseng storage root among plant storage tissues, the ginseng tissue must undergo a process for dedifferentiation of the storage root (differentiated tissue) into undifferentiated tissue, in any kind of production method among currently known methods. In this process, the somaclonal variation may unavoidably occur. In other words, in order to produce the cells of ginsengs in large amounts using plant tissue culture techniques, a genetically stable sample must be used as a material in order to reduce somaclonal variation. It was reported in Korean Patent Publication 10-2005-0078372 that somaclonal variation basically occurs even if any tissue of ginsengs is used.
Meanwhile, cambium is a tissue that thickens the stem and root to allow the plant grow volumetrically. It was reported that when the cambium, a meristem where the most active cell division occurs, is used as an explant for plant cell tissue culture, rapid and mass production of cells is possible (Korean Patent Registration 10-0533120). Studies on structure and ultrastructure of this cambium have progressed slowly because of inherent technical difficulty in using the material. It was reported that, because the cambium is composed of several narrow, elongated and thin-walled cell layers, it is easily damaged during extraction. Also, it was reported that highly vacuolated active meristematic cells are difficult to fix even either by a conventional method employing an electron microscope or by techniques recently developed in order to study the in situ localization of proteins, RNAs and other molecules (Lachaud Suzanne et al., Life Science, 633, 1999).
In addition, the mechanical sectioning of the continuous cambium was not widely used, and this is believed to be because of the technical difficulties of isolating cambium cells that have long length and are thin-walled. In many studies, it was reported that the shape, size and arrangement of cambium cells were characterized indirectly based on the structure of cambium derivatives on the assumption that the structure of the secondary vascular tissue reflects the cambium (Kitin, P. et al., Ann. Bot., 86:1109, 2000). In other words, several studies suggest that there is much difficulty in using cambium directly as a material for studies in various fields.
Korean Patent Registration 10-0533120 developed by some of the present inventors discloses a method of inducing callus using the cambium collected from the stem of a plant. This registered patent relates to a plant cell culture method for obtaining plant cells rapidly in large amounts and mentions a plant cell culture method of inducing callus through the cambium collected from the plant stem rather than using a general seed culture method. The registered patent suggests a method of inducing cambium cells by using the cambium of woody plant stem with addition of high concentrations of auxin picloram and gibberellic acid, but in this registered patent, the callus is merely induced from the cambium of woody plant stem. Because the callus is a tissue formed through a dedifferentiation process, this registered patent still has the problem of variation caused by dedifferentiation.
Furthermore, some of the present inventors developed the invention of PCT/KR 2006/001544, which solves the problem of variation caused by dedifferentiation and relates to a method for providing cell lines that can stably proliferate and have high genetic stability. The method disclosed in the PCT application also uses the cambium of woody plant stem, but because morphological and physiological characteristics of herbaceous plants such as ginseng plants are different from those of woody plants, there has been a need to develop an improved invention which considers the characteristics of herbaceous plants in order to induce cell lines from the cambium of storage root tissue of herbaceous plants.
Accordingly, the present inventors have made extensive efforts to obtain a plant cell line, which is a homogeneous cell line having the ability to divide, and has not undergone a dedifferentiation process, and thus has no somaclonal variation during culture. As a result, the present inventors have isolated a cambium-derived cell line by applying osmotic stress to cambium-containing storage root tissue and culturing the storage root tissue in a specific plant hormone-containing medium, and have found that the isolated cell line is a homogeneous cell line, which has an unlimited ability to divide, has been isolated without a dedifferentiation process to have no somaclonal variation, and thus is genetically highly stable and physiologically uniform, thereby completing the present invention.